a) Field of the Invention
The invention is directed to a device for coupling light into the reflected light beam path of a microscope. It is applicable in particular for implementing the known microscopy method of total internal reflection fluorescence (TIRF) in which light is radiated into the specimen or preparation to be examined at an angle greater than the angle of total reflection at the interface or boundary layer between the coverslip and the preparation. Through total reflection, the preparation is illuminated by an evanescent field which only has a penetration depth of 100–200 nm. Therefore, fluorescence excitation can only come about in this range and during detection of the fluorescence signal the signal-to-noise ratio can be improved over conventional methods.
b) Description of the Related Art
This is used, for example, to examine intracellular and intercellular transport processes at cell membranes, etc., located directly on the surface of the coverslip.
The first experiments were described by Daniel Axelrod, et al.: Stout and Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy”, Dec. 1989, Applied Optics, Vol. 28, No. 24, page 5237; Sund, Swanson and Axelrod, “Cell Membrane Orientation Visualized by Polarized Total Internal Reflection Fluorescence”, Oct. 1999, Biophysical Journal, Vol. 77. Additional references include Zenisek, Steyer and Almers, “Transport, capture and exocytosis of single vesicles at active zones”, Aug. 2000, Nature, Vol. 406.
In principle, there are two possibilities for carrying out TIRF microscopy: with transmitted light and with reflected light.
a) In the transmitted light variant, the condenser must be replaced by a laser in-coupling prism system such as is known, e.g., from DE 199 23 563 A1 of Dec. 7, 2000. This solution has a number of disadvantages. It is unsuitable for inverted microscopes because the space required over the preparation in these microscopes is hidden by the prism, so that access to the preparation which is needed for many experiments is blocked.
b) The reflected light variant requires an objective with a sufficiently high numerical aperture on the one hand and a coupling in of the laser through this objective on the other hand. In known solutions, the laser is coupled in through the epi-fluorescence beam path and requires that the standard reflected illumination be replaced by a special version, or a beam path parallel to the standard reflected illumination is used for a TIRF laser in-coupling. A solution of this kind is produced by the firm T.I.L.L. Photonics under the trade name TILL-TIRF Module. Exchanging the entire reflected illumination apparatus makes the system expensive and inflexible.